Synthesis of 4’,5,7-trihydroxyflavanone and 3’,4’,5,7-tetrahydroxyflavanone and antioxidant activity

Natural flavonoids, 4’,5,7-trihydroxyflavanone and 3’,4’,5,7-tetrahydroxyflavanone were synthesised via their respective chalcone. The initial step was to synthesise derivatives of 2-hydroxyacetophenone and benzaldehyde by protecting the phenolic hydroxyl groups. The chalcone was synthesised by Claisen-Schmidt condensation. Acid hydrolysis and subsequent treatment with sodium acetate of 2’-hydroxy-4,4’-6’-tris(methoxymethyloxy)chalcone and 2’-hydroxy-3,4,4’,6’-tetrakis(methoxymethyloxy)chalcone, gave 4’,5,7-trihydroxyflavanone and 3’,4’,5,7-tetrahydroxyflavanone, respectively. 3’,4’,5,7-Tetrahydroxyflavanone was found to be more potent as an antioxidant agent than 4’,5,7-trihydroxyflavanone with 83.11% inhibition and SC50 8.57 μg/mL in the radical scavenging activity by ESR method. | 4’,5,7-Trihydroxyflavanone | 3’,4’,5,7-Tetrahydroxyflavanone | Chalcones | Antioxidant | ESR | ® 2010 Ibnu Sina Institute. All rights reserved.


INTRODUCTION
Flavonoids are a large group of polyphenolic compounds possessing a basic flavan nucleus with two aromatic rings (the A and B rings) interconnected by a three-carbon-atom heterocyclic ring (the C ring). The most widespread flavonoids contain a double bond between C-2 and C-3 and a keto function at C-4 of ring C [1]. Flavonoids represent one of the largest and most diverse classes of plant secondary metabolites which are naturally present in vegetables, fruits, and beverages. They possess a wide variety of biological activities including antiinflammatory, antioxidant, antimutagenic, anti-HIV, vasodilator, anticancer, and cardiovascular effects [2]. The antioxidant activities of flavonoids have been evaluated against reactive oxygen species like 1,1-diphenyl-2picrylhydrazyl (DPPH) radical using the UV technique [3]. The application of the Electron Spin Resonance (ESR) technique to test the direct scavenging activities of flavonoids was first published by Goa et al. in 1999 [4].
The interest to study the antioxidant activity of the flavonoids has prompted us to synthesise naringenin (1) and eriodictyol (2); two hydroxylated flavanones abundant in grape fruit and lemon, respectively [5,6].

Materials, method and instruments
Melting points were recorded on a Leica Galen III Kofler micro melting point apparatus and were uncorrected. Infrared (IR) spectra were recorded on Shimadzu 8000 or Perkin-Elmer series 1600 spectrometers as thin film for liquid samples or KBr pellet for solid samples. Mass spectral data were obtained from Kent Mass Spectrometry Service, UK. 1 H and 13 C NMR spectra (300 and 75 MHz, respectively) were recorded on a Bruker Avance 300 Spectrometer using CDCl 3 and DMSO as solvent. Reactions were monitored by thin-layer chromatography (tlc) carried out on 0.2 mm Merck pre-coated silica gel plates (60 F 254 ).

Antioxidant screening (free radical scavenging activity)
DPPH radical scavenging using ESR (Electron Spin Resonance) was carried out according to the method described by Ohtani et al. [10] with some minor modifications. The ethanolic solution of the test sample 200 µL (1mg/mL) was added to 200 µL of DPPH (0.25 mM) in ethanol solution. After shaking vigorously for 10 sec, the solution was transferred to a flat cell. The ESR spectra were recorded after 40 sec. The condition of ESR spectrometer were set at room temperature, power 1mW, magnetic field 336.000 ± 5mT, field modulation width 0.5 mT, sweep time 30 sec and time constant 0.03 sec. The scavenging effect of DPPH was calculated by the following formula: PH DPPH-PH Sample Percent Scavenging (%) = x 100 PH DPPH PH= Peak height of the third and the fifth line signals of DPPH radical.
The SC 50 value was determined as the concentration of each sample required to give 50% of scavenging of DPPH. All test and analyses were run in triplicates.
Deprotection of the MOM group of chalcone (9) was achieved by using HCl (10%) in MeOH and cyclisation was carried out in NaOAc/MeOH to furnish (1) as brown solids in 85.1% yield. The IR spectrum displayed a broad absorption band at 3300-3200 cm -1 attributable to the hydroxyl group. The UV spectrum showed characteristics absorption for a flavanone at 330 (band I) and 289 (band II) nm. A bathochromic shift of 35 nm was noted for band II after an addition of NaOH and NaOAc shift reagents, indicating a 5,7-OH flavanone [12]. The CIMS spectrum showed a molecular ion peak at m/z 272 which was in accordance with the molecular formula C 15 H 12 O 5 . The 1 H NMR spectrum displayed typical ABX splitting pattern for a flavanone at  H-4'). The IR spectrum displayed stretching bands for -OH at 3443 cm -1 and a carbonyl at 1623 cm -1 . The 1 H NMR spectrum of (8) showed the presence of signals due to methylene protons at δ 3.47 (3H) and 3.53 (3H), which proved that (7) has been protected. The phenolic proton resonated as a singlet at δ 13.71. (c) i. HCl 10%, MeOH; ii. NaOAc, reflux 3 h.

Electron Spin Resonance (ESR) spectrometry method
In this study, the ESR spectrometry assay has been used to measure the free radical scavenging activity of the antioxidant against DPPH radical. Vitamin C was used as a reference antioxidant and screened for comparison purposes with the synthesised flavanones. Positive DPPH test suggests that the tested compounds are free radical scavengers.
An ESR signal is directly proportional to the number of radicals present. The 0.25 mM DPPH radicals give a typical ESR spectrum as shown in Figure 1.0. The peaks height will be reduced when an antioxidant was added to the ethanolic DPPH solution. The radical scavenging activity of flavanones was expressed by means of SC 50 which represent the amount of antioxidant necessary to decrease the initial DPPH concentration by 50%.   Table 1.0 shows the activity of the tested compounds. The radical scavenging activity of the active compounds was found to be concentration-dependent manner. The data reveal that 3',4',5,7-tetrahydroxyflavanone (2) possess the strongest activity as free radical scavenger compared with positive control, vitamin C and flavanone (1), which can be seen from the SC 50 value of this compound; 8.57 µg/mL and 83.11% inhibition at concentration 15.63 µg/mL. Figure 1.0 shows the intensities of the DPPH signal at concentration of 15.63 µg/mL for the flavanoids and vitamin C.

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Flavonoids with free hydroxyl groups are known to scavenge free radicals by hydrogen donation. From the results obtained above, the order of effectiveness in scavenging DPPH radicals, also a measure of their antioxidative potentials, is as follows: 3',4',5,7-tetrahydroxyflavanone (2) > vitamin C > 4',5,7trihydroxyflavanone (1) An increase in the number of hydroxyl groups generally enhances the antioxidant activity of the flavonoids [14]. 3',4',5,7-Tetrahydroxyflavanone (2) which possess 3',4'-dihydroxyl group in B-ring showed higher antioxidant activity than 4',5,7-trihydroxyflavanone (1). The B-ring which has the hydroxyl configuration is the most important determinant of scavenging free radical. It was found that flavonoids with the presence of catechol group in ring B was essential for high antioxidant activity. 4',5,7-Trihydroxyflavanone (1) which lacked the orthodihydroxylation in the B ring reduced the radical scavenging activity.